Summary of Research in Progress[unreadable] [unreadable] Currently, there are two main ongoing projects in the lab:[unreadable] [unreadable] The first project is focused on elucidating mechanistic details of the interaction between type II topoisomerases and DNA. One aspect of this interaction concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that these enzymes preferentially unlink rather than link DNA. However the mechanism by which an enzyme that acts locally on the scale of nanometers can determine the global linking topology of micron sized DNA molecules remains a mystery. One proposed mechanism suggests that unlinking may be favored over linking if the topoisomerase induces a sharp bend in the DNA on binding. We are currently using atomic force microscopy to directly image type II topoisomerases bound to DNA. From these measurements we hope to extract the induced bend angle, which in combination with Monte Carlo simulations of DNA molecules with a given bend angle, will allow us to determine if the topoisomerase induced bending model can explain the observed unlinking/linking asymmetry.[unreadable] [unreadable] A second aspect of the interaction between type II topoisomerases and their DNA substrates concerns the diverse topological activities exhibited by type II topoisomerases that share a common reaction mechanism. Specifically, we are studying the molecular basis underlying the range of activities catalyzed by different isoforms of type II topoisomerases. These activities include the symmetric relaxation of positively and negatively supercoiled DNA by most type II topoisomerases, the introduction of negative supercoils by DNA gyrase, and the asymmetric relaxation of negative and positive supercoils by some type II enzymes. These differences in activity are believed to arise from differences in the poorly conserved C-terminal domains (CTDs), but the molecular basis underling these variations in activity have not been elucidated. We have produced a series of specific mutations in the CTD region of Topoisomerase IV. We are employing a combination of ensemble and single molecule assays to test the effects of these mutations on the substrate selectivity and processivity of topoisomerase IV. We are also investigating the role of the CTD linker on the activity of Topoisomerase IV. [unreadable] [unreadable] The second project is focused on extending single-molecule techniques to dissect the detailed mechanisms underlying multi-enzyme complex formation and activity. Helicases of the RecQ family and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of the interaction between the two enzymes leads to severe chromosome instability, however the mechanisms underlying their interaction, and the specific activity of the coupled enzyme remain unclear. Analysis of the coupled enzyme system is complicated by the fact that both the helicase and the topoisomerase individually modify the structure of DNA, and these activities must be distinguished from the activity of the coupled enzymes. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. Following the activity of a single enzyme or multi-enzyme complex over time can reveal transient phenomena, fluctuations in activity, and the presence of enzyme sub-populations or enzymatic states, all of which are obscured by the averaging inherent in traditional ensemble measurements. In one project, we are investigating the activity of reverse gyrase, a unique topoisomerase from hyperthermophilic bacteria that is comprised of a helicase and a topoisomerase on a single polypeptide. Reverse gyrase serves as a model system in which to study the interaction of a helicase and a topoisomerase. Through the concerted activity of the two domains, reverse gyrase promotes the positive supercoiling (over-winding) of DNA, however the mechanism underlying this activity remains speculative. Single-molecule experiments will allow us to probe the details of the supercoiling reaction, and in conjunction with non-hydrolysable ATP analogs and point mutations, will allow us to determine the molecular basis for communication between the helicase and topoisomerase domains. In the second project are using single-molecule fluorescence techniques, primarily fluorescence resonance energy transfer (FRET), to measure the binding kinetics of RecQ helicase and Topo III form E. coli in isolation and in the presence of a variety of DNA substrates and nucleotide cofactors. These experiments and the experimental techniques employed will pave the way for more complex experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques. [unreadable] [unreadable] Future research goals:[unreadable] [unreadable] Our immediate goal is the completion of the ongoing projects in the lab. Longer term goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection capabilities. This instrument will permit simultaneous measurements of the activity and composition of multi-enzyme complexes interacting with a single DNA molecule. The activity of the complex can be determined from the mechanical changes in the DNA, or from the motion of the complex along the DNA strand. The composition of the complex can be determined from multicolor fluorescence detection and localization. This instrument will open up other areas of research including the possibility of observing the dynamics of supercoiled DNA.